The invention relates to a method for generating an artificially patterned substrate for stimulating the crystallation of a biomolecule. Further, the invention relates to a method for stimulating the crystallization of biomolecules from a liquid solution containing said biomolecules.
Generally, collecting information related to the structure of proteins and other biomolecules is becoming an important field of technology. With information on the three-dimensional structure of biomolecules, knowledge on many fundamental processes of life can be deepened and the working mechanisms of essential biological processes can be partially or fully elucidated. This knowledge may allow for the more efficient synthesis of bio-active compounds and the optimization of the development of pharmaceuticals.
For this reason, the determination of molecular structures of proteins is an immensely important task in biological research. The most common method so far for structure determination is X-ray diffraction, which requires proteins to be crystallized before they can be analysed. Protein crystals are often obtained only after lengthy and labour-intensive screening processes. A considerable number of proteins resist crystallization efforts or yield crystals of poor quality which are not suitable for structure determination. Current laboratory practices are largely based on empirical results rather than on a fundamental understanding of crystal nucleation and growth at a physical level.
In the under the Patent Cooperation Treaty (PCT) published international patent application WO 01/92293 A2 are methods for nano-crystallogenesis disclosed. The disclosure relates so far to methods for identifying crystallization conditions for biomolecules using e.g. a method and an apparatus for screening the phase behaviour in liquid gel or solid phase of such biomolecules, like proteins, peptides, other macromolecules or complexes thereof. This publication also discloses methods for the screening of crystallization conditions and for allowing crystals with higher quality to be grown. These results are achieved by establishing a method for identifying crystallization conditions for at least one biomolecule. This method subjects said biomolecule to a set of compositions in a sample volume of 1 to 100 nanoliters in order to induce or to allow each of said compositions to adopt at least one first condition possibly allowing said crystallization to occur and to detect whether crystallization has occurred or not. The screening disclosed in this publication is performed by differing the test conditions from each test volume to the next. An appropriate method for the preparation of a solid support medium containing wells with a volume of up to 100 nanoliter provides a layer of negative photoresist on a carrier body and applies a pattern to said photoresist. Afterwards UV-illumination is applied to the photoresist and then the non-illuminated parts of the photoresist are removed.
The main drawback of this disclosure is that the methods proposed do not give any contribution to a better fundamental understanding of the crystallization and growth of proteins and other macromolecules. These methods are based more or less on arbitrary screening approaches to explore the optimal condition for the crystallization of biomolecules. It has to be mentioned that this lack of awareness for the fundamentals of macromolecular crystallization is inherent to the current state of art in this field of research.
One of the most promising attempts in the last years has been published by X. M. Yang, R. D. Peters, P. F. Nealey, H. H. Solak, F. Cerrina, “Guided self assembly of Symmetric Diblock Copolymer films on chemically nanopatterned substrates, Macromolecules 33, p. 9575. This publication recently demonstrated the organization of macromolecules by chemical contrast of the surface of a substrate matching the natural periodicity of the molecular structures by guided self-assembly of block copolymers. Block copolymers naturally form periodic structures due to micro-phase forces. The resultant structures are not crystalline but they are both regular and periodic. In this study a Self Assembled Monolayer (SAM) coated Si wafer was subjected to X-ray interference lithography (XIL) to obtain a periodic surface structure with chemical contrast. The block copolymer was then deposited on this template surface and annealed to activate the micro-phase separation process. The block copolymers were observed to arrange their domains according to the artificial template structure which was designed to match the natural periodicity of the copolymer.
Unfortunately, this technology does not apply to the stimulation of the crystallization of biomolecules since the attraction of copolymer self assembly is induced by micro-phase forces and is supported by the fact that a sufficient amount of the material to be self assembled is available. Very often in the field of biomolecular research the determination of the conditions under which crystallogenesis of biomolecules is efficient suffers from another drawback, namely that the available amounts of the target biomolecules are severely limited. More often than not biomolecules are available only in minute quantities (micrograms or nanograms), generally obtained after long and tedious processes.